Protein C (PC) is a member of the class of a vitamin K-dependent serine protease coagulation factors. Unlike the majority of coagulation factors, Protein C regulates blood coagulation by acting an a natural anticoagulant that circulates in the blood in an inactive form that requires proteolytic activation to generate the anticoagulant enzyme. The activated form of Protein C, APC, inhibits blood coagulation at the levels of Factor V and VIII in the clotting cascade.
Similar to most other zymogens of extracellular proteases, Protein C has the core structure of the chymotrypsin family having insertions and N-terminus extensions that enable regulation of the zymogen and the enzyme. See Owen W., in "Hemostasis and Thrombosis: Basic Principles and Clinical Practice", Colman et al., eds, pp. 235-241, J. B. Lippincott Co. (Philadelphia), 1987. The isolated zymogen from human consists of two polypeptide chains, a heavy and a light chain having the respective molecular weights of 41,000 and 21,000 daltons, linked by a single disulfide. The light chain is 155 amino acid residues in length, the first 11 glutamate residues of which reside within the first 35 amino acid residues and are gamma-carboxylated. The zymogen heavy chain is 262 amino acid residues long which is cleaved to a final 250 amino acid residues in length upon activation.
Protein C is composed of domains with discrete structure and function. See Foster et al., Proc. Natl. Acad. Sci. USA, 82:4673-4677 (1985) and Plutzky et al., Proc. Natl. Acad. Sci. USA, 83:546-550 (1986). The light chain contains an animo-terminal gamma-carboxyglutamic acid (Gla) region which is followed by two domains that are homologous to domains in the epidermal growth factor (EGF) precursor. The serine protease activity resides in the heavy chain. Ohlin et al., Biochem., 29:644-651 (1990).
The zymogen is activated by the action of thrombin at the site between the arginine residue at position number 12 and the leucine residue at position 13. See Kisiel, J. Clin. Invest., 64:761-769, (1976); Marlar et al., Blood, 59:1067-1072 (1982). Other proteins including Factor Xa (Haley et al., J. Biol. Chem., 264:16303-16310 (1989), Russell's viper venom and trypsin (Esmon et al., J. Biol. Chem., 251:2770-2776 (1976) have also been shown of enzymatically cleave inactive protein C to its activated form. Activated protein C hydrolyzes arginine esters and related substrates via a core triad of catalytic amino acid residues which occur at Ser-201, His-56, and Asp-102 of the heavy chain. The triad forms a hydrophobic substrate binding pocket. The enzyme's specificity is restricted to a small number of protein substrates; blood coagulation cofactors, activated Factors V and VIII, are the only known macromolecular substrates for the proteolytic inactivation by activated protein C. See Kisiel et al., Biochem., 16:5824-5831 (1977); Vehar et al., Biochem., 19:401-410 (1980); and Walker et al., Biochim. Biophys. Acta., 571:333-342 (1979).
Thrombin, the major physiological protein C activator, activates protein C slowly in purified systems, plasma, or blood, when in the presence of physiolgical concentrations of calcium. A membrane-bound thrombin receptor called thrombomodulin has been identified which accelerates protein C activation. Esmon et al., Proc. Natl. Acad. Sci. USA, 78:2249-2252 (1981). Liberated thrombin binds to thrombomodulin on the luminal surface of endothelial cells and undergoes an increase in specificity for circulating protein C. Calcium is required for this process and is bound to calcium-binding domains in the EGF-like regions of protein C. Additional studies have revealed that the membrane-lipid domain of protein C, the vitamin-K dependent Gla domain, is also required for activation of protein C. Esmon et al., in "Progress in Vascular Biology, Hemostasis, and Thrombosis", Ruggeri et al., eds., Annals of The New York Academy of Sciences, Vol. 614:30-43 (1991).
Inhibitors of activated protein C which function to inhibit anticoagulant activity have been characterized. Malar and Griffin characterized a partially purified inhibitor in plasma, the binding of which to activated protein C results in the complete loss of anticoagulant and esterolytic activities. Marlar et al., J. Clin. Invest., 66:1186-1193 (1980). Suzuki and collaborators have purified the heparin-dependent inhibitor and determined that the inhibition of the activated enzyme occurred through the formation of an enzyme-inhibitor complex accompanied with the proteolytic modification of the inhibitor by the enzyme. Suzuki et al., J. Biochem., 95:187-195 (1984). More recently, a heparin-independent inhibitor of activated protein C has been characterized. Heeb et al., J. Biol. Chem., 265:2365-2369 (1990). The active site of this enzyme called .alpha..sub.1 -antitrypsin occurs at the methionine residue at position 358 the binding of which results in the inactivation of activated protein C. Patients with a combined deficiency of Factor V and Factor VIII have been shown to also lack the heparin-dependent form of inhibitor. Marlar et al., supra, (1980).
Clinical evidence for a regulatory role of protein C as well as its inhibitors is abundant. A family having a protein C deficiency was identified in 1981 having the symptoms of episodes of severe recurrent venous thrombosis. Patients having abnormal protein C which cannot be activated by the thrombin-thrombomodulin complex have also been identified. Comp et al., Blood, 63:15-21 (1984); Griffin et al., Blood, 62:301a (1983); and Sala et al., Blood, 63:671-675 (1984).
Neonatal purpura fulminans is a condition in newborn infants who are homozygous for deficiency of protein C and consequently, develop severe clotting in the capillaries of the skin. Branson et al., Lancet, 2:1165-1168 (1983); Seligsohn et al., N. Eng. J. Med., 310:559-562 (1984). In view of the properties of activated protein C, a deficiency of the enzyme might result in a hypercoaguable state.
One advantage of this condition is that coagulation cofactors V and VIII, which are normally inactivated by activated protein C, remain in an activated state. Methods for purifying Factor V and Factor VIII are well known in the art. See, for example, U.S. Pat. Re. No. 32,001; EPO Publication No. 160,457; Vehar et al., supra; Dahlback et al., J. Clin. Invest., 66:583-591 (1980); and Suzuki et al., J. Biol. Chem., 257:6556-6564 (1982). Methods for purifying the activated forms of Factor V and Factor VIII are not available as the activated factors are rapidly degraded in the blood under normal conditions by activated protein C.
In the present invention, synthetic polypeptides corresponding to the heavy chain domain of protein C have been discovered which have the ability to inhibit the anticoagulant properties of activated protein C. The present invention, thus, contemplates a method for purifying Factor Va and Factor VIIIa through the use of an activated protein C-inhibiting amount of a composition comprising a protein C derived synthetic polypeptide, antibody, or a monoclonal antibody. The method is based on the discovery described herein of inhibitors of activated protein C and their demonstrated utility in inhibiting the enzyme in vitro.
No polypeptides having this activity have ever been described before this present invention. In fact, protein C from bovine has only been proteolytically cleaved into two fragments. The larger one consists of the entire light chain of protein C having two epidermal growth factor-like domains and the Gla domain along with 23 heavy chain residues. Ohlin et al., supra. This fragment exhibits inhibitory activity towards activated protein C but it lacks the regions defined in the present invention which specify the smallest peptide which inhibits activated protein C.